Project Summary Identification of antigens that are involved in the pathogenesis of type 1 diabetes (T1D) is crucial to develop biomarkers and therapies for T1D. There have been recent efforts focused on identifying antigens recognized by T cells within the residual islets of T1D organ donors due to the premise that such T cells are likely to be involved in disease pathogenesis. Our preliminary data studying islet-infiltrating CD8 T cells demonstrates that a large proportion, 1/3 to 1/2 of T1D organ donors, are reactive to preproinsulin (PPI) peptides, whereas the remaining donors have only few or no PPI-reactive CD8 T cells in the islets. Epitopes recognized by the PPI- reactive CD8 T cells are presented by many HLA class I molecules spanning HLA-A, B, and C, and are spread throughout the PPI protein with several hot spots that are preferentially recognized by multiple T cell receptors (TCR) expressed by the T cells. Thus, preproinsulin is a major self-antigen and contains epitopes for T1D- associated CD8 T cells in a subset of T1D patients. Yet antigens targeted by islet-infiltrating T cells in the remaining patients exist outside PPI, suggesting heterogeneity in antigen specificity targeted by islet-resident CD8 T cells. Towards an ultimate goal of developing antigen-specific biomarkers and therapies for T1D, two important questions remain; (1) What antigens are recognized by islet-infiltrating CD8 T cells that do not react with PPI? (2) By measuring reactivity in peripheral blood, can we identify patients that have a particular antigen specificity in order to design appropriate therapy for each patient? The goal of this proposal is to uncover unknown antigen specificities recognized by islet-derived CD8 T cells and to evaluate the association between PPI reactivity in peripheral blood and the response to oral insulin as a proof of principle for personalized antigen- specific therapies. Our central hypothesis is that heterogeneity in self-antigen specificity for islet-derived CD8 T cells exists and determines the response to antigen-specific immunotherapy. If this hypothesis is correct, self- antigens targeted by CD8 T cells can be utilized to identify patients with particular antigen specificity to select for responders receiving antigen-specific immunotherapies for T1D prevention efforts. To test the hypothesis, we will identify antigens that are recognized by islet-infiltrating CD8 T cells either in a protein-targeted manner (Aim 1) or in an unbiased manner (Aim 2). Given our preliminary results that PPI is a major antigen for islet-infiltrating CD8 T cells in a subset of patients, we will focus on PPI to test whether there is an association between CD8 PPI reactivity and the response to oral insulin treatment (Aim 3). The successful completion of this proposal will results in identification of major self-antigens for islet-resident CD8 T cells in T1D patients. The identification of antigens can be used to classify heterogeneous T1D patients based on CD8 T cell antigen specificity with the goal to ultimately providing appropriate personalized antigen-specific immunotherapy for T1D prevention efforts.